A New Era for Cancer Immunotherapy Based on the Genes that Encode Cancer Antigens

Abstract

In 1929, reviewing the available information concerning cancer immunotherapy, W. H. Woglom wrote, "It would be as difficult to reject the right ear and leave the left ear intact as it is to immunize against cancer" (1929). Minimal progress had been made since the earliest descriptions of attempts to immunize against cancer by Nooth, the surgeon to the Duke of Kent, who in 1777 inoculated himself with cancer tissue, or the physician to Louis XVIII, who in 1808 injected himself with breast cancer tissue. Knowledge of the cellular immune system was sparse, and as late as 1958 the Journal of Immunology did not list the word "lymphocyte" in its index. Most attempts to develop cancer vaccines involved immunization of cancer patients using either their own or allogeneic tumors along with a variety of nonspecific immune adjuvants. Even as the basic tenets of modern cellular immunology were elucidated, studies of cancer immunotherapy languished at the periphery of respectable science due to a lack of information regarding the molecular identification of the components of the putative immune reaction against human cancers. In the past decade, however, the convergence of information from basic studies of cellular and molecular immunology and the application of recombinant DNA techniques to produce pharmacologic quantities of biologic molecules normally present only in minute amounts have substantially changed views concerning the immune response to human cancer and have provided the first demonstrations that immune reactions against cancer antigens can lead to the regression of invasive tumors in selected patients. The molecular identification of tumor antigens, their immunodominant peptides, and the T cell receptors that recognize them have placed studies of tumor immunology and immunotherapy in the mainstream of immunologic research. Four major techniques have been used to identify cancer antigens capable of eliciting cellular immune reactions in humans. The majority of human tumor antigens now known have been identified by the transfection of genomic DNA or cDNA libraries into cells expressing the appropriate MHC molecule, followed by the identification of transfectants using cytokine release or lysis by human T cells with specific antitumor reactivity (2Boon T. Tumor antigens recognized by cytolytic T lymphocytes present perspectives for specific immunotherapy.Int. J. Cancer. 1993; 54: 177-180Crossref PubMed Scopus (97) Google Scholar, 43Rosenberg S.A. Development of cancer immunotherapies based on identification of the genes encoding cancer regression antigens.J. Natl. Cancer Inst. 1996; 88: 1635-1644Crossref PubMed Scopus (120) Google Scholar). Biochemical approaches have had more limited success in the identification of human cancer antigens. Attempts have been made to elute peptides from tumor cells or from MHC molecules purified from tumor cells and to detect fractions capable of stimulating antitumor T cells after pulsing purified fractions onto antigen-presenting cells. Triple quadrapole mass spectrometric techniques have then been used to sequence the minute quantities of peptides obtained (11Cox A.L. Skipper J. Chen Y. Henderson R.A. Darrow T.L. Shabanowitz J. Engelhard V.H. Hunt D.F. Slingluff C.L. Identification of a peptide recognized by five melanoma-specific human cytotoxic T cell lines.Science. 1994; 264: 716-719Crossref PubMed Scopus (799) Google Scholar). This approach has been severely limited by the need for custom-made, highly specialized equipment and the requirement that peptides be present in sufficient quantity to enable their identification by these physical techniques. The ability of antigen-presenting cells to endocytose proteins and present peptides on class II MHC molecules has been utilized to identify antigens recognized by CD4 T cells, although thus far few antigens have been identified using this approach (61Topalian S.L. Rivoltini L. Mancini M. Markus N.R. Robbins P.F. Kawakami Y. Rosenberg S.A. Human CD4+ T cells specifically recognize a shared melanoma-associated antigen encoded by the tyrosinase gene.Proc. Natl. Acad. Sci. USA. 1994; 91: 9461-9465Crossref PubMed Scopus (263) Google Scholar). Each of the three techniques mentioned above is dependent on the prior availability of T cells capable of recognizing tumor antigens, a requirement that often cannot be met. A fourth approach to the identification of tumor antigens has involved attempts to develop, by in vitro sensitization techniques, T cells against candidate tumor antigens (37Parkhurst M.R. Fitzgerald E.B. Southwood S. Sette A. Rosenberg S.A. Kawakami Y. Identification of a shared HLA-A*0201-restricted T-cell epitope from the melanoma antigen tyrosinase-related protein 2 (TRP2).Cancer Res. 1998; 58: 4895-4901PubMed Google Scholar). T cells successfully generated in vitro against candidate antigens have then been tested for their ability to recognize intact tumor cells, and the presence of such reactivity provides strong evidence that these candidate proteins represent tumor antigens. Genes encoding candidate tumor antigens have been transfected or transduced into antigen-presenting cells or synthesized peptides from candidate antigens based on known MHC-binding motifs have been pulsed onto antigen-presenting cells and used for these in vitro sensitizations. An alternate technique for the identification of tumor antigens that is also not dependent on the prior availability of antitumor T cells has recently been described. This approach, called serological analysis of recombinant cDNA expression libraries (SEREX), uses diluted serum from cancer patients to detect prokaryotically expressed cDNA libraries prepared from tumors (51Sahin U. Tureci O. Schmitt H. Cochlovius B. Johannes T. Schmits R. Stenner F. Luo G. Schobert I. Pfreundschuh M. Human neoplasms elicit multiple specific immune responses in the autologous host.Proc. Natl. Acad. Sci. USA. 1995; 92: 11810-11813Crossref PubMed Scopus (955) Google Scholar, 8Chen Y.T. Scanlan M.J. Sahin U. A testicular antigen aberrantly expressed in human cancers detected by autologous antibody screening.Proc. Natl. Acad. Sci. USA. 1997; 94: 1914-1918Crossref PubMed Scopus (1051) Google Scholar). This approach is based on the assumption that antibody production implies that a helper T cell reaction exists against the detected antigen. At least one tumor antigen, NY-ESO-1, subsequently shown to be reactive with CD8+ T cells, has been identified using this approach (8Chen Y.T. Scanlan M.J. Sahin U. A testicular antigen aberrantly expressed in human cancers detected by autologous antibody screening.Proc. Natl. Acad. Sci. USA. 1997; 94: 1914-1918Crossref PubMed Scopus (1051) Google Scholar), and other candidate antigens are being studied (65Tureci O. Sahin U. Schobert I. Koslowski M. Schmitt H. Schild H.-J. Stenner F. Seizt G. Rammensee H.-G. Pfreundschuh M. The SSX-2 gene, which is involved in the t(X;18) translocation of synovial sarcomas, codes for the human tumor antigen HOM-MEL-40.Cancer Res. 1996; 56: 4766-4772PubMed Google Scholar). An unexpected diversity of mechanisms can result in the generation of antigenic epitopes recognized by tumor-specific T cells. These epitopes have come from normal nonmutated genes whose expression is limited to cancer and selected normal tissues, from aberrantly expressed intronic sequences, from alternative open reading frames of normal genes, and from mutations specific to the individual cancer (Table 1).Table 1Defined Cancer AntigensAntigen CategoryGeneMHC RestrictionMelanocyteMART-1/MelanAA2, B45differentiationgp100A2, A3, A24TyrosinaseA1, A2, A24, DR4TRP-1A31TRP-2A2, A31, A68Cancer testisMAGE-1A1, Cw16MAGE-3A1, A2, B44GAGE-1/2Cw16BAGECw16RAGEB7NY-ESO-1A2, A31Tumor specificCDK-4A2β-cateninA24MUM-1B44Caspase-8B35KIAA0205B44HPVE7A2Widely expressedSART-1A26PRAMEA24p15A24 Open table in a new tab Malignant melanomas have been a particularly rich source of tumor antigens that are found not only on melanomas but on other tumor types as well. Two major categories of normal, non-mutated genes that encode shared tumor antigens have been identified: (1) differentiation antigens shared on melanomas and melanocytes, and (2) differentiation antigens shared on a variety of tumors as well as normal testes. The expression of one family of differentiation antigens is limited to melanomas as well as melanocytes, the cell of origin of this tumor, and pigment-producing cells in the retina. The T cells used to identify these melanoma/melanocyte differentiation antigens have largely been derived from tumor-infiltrating lymphocytes (TIL) obtained from patients with growing cancers or from autologous mixed-lymphocyte tumor cultures (2Boon T. Tumor antigens recognized by cytolytic T lymphocytes present perspectives for specific immunotherapy.Int. J. Cancer. 1993; 54: 177-180Crossref PubMed Scopus (97) Google Scholar, 43Rosenberg S.A. Development of cancer immunotherapies based on identification of the genes encoding cancer regression antigens.J. Natl. Cancer Inst. 1996; 88: 1635-1644Crossref PubMed Scopus (120) Google Scholar). The presence of precursors capable of reacting with normal nonmutated self-antigens within the tumor has suggested that the inflammatory microenvironment at the site of the tumor has resulted in the breaking of peripheral tolerance to these antigens. Alternatively, the tumor may have acted as a "sink" for the accumulation of precursors normally present, since precursors to at least one of these antigens has also been found in normal individuals. The most common antitumor reactivity found in melanoma patients is directed against the MART-1/MelanA antigen, which is a 118–amino acid protein of unknown function that contains a 21–amino acid transmembrane region (9Coulie P. Brichard V. Van Pel A. Wolfel T. Schneider J. Traversari C. Mattei S. DePlaen E. Lurquin C. Szikora J. et al.A new gene coding for a differentiation antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas.J. Exp. Med. 1994; 180: 35-42Crossref PubMed Scopus (857) Google Scholar, 20Kawakami Y. Eliyahu S. Delgado C.H. Robbins P.F. Rivoltini L. Topalian S.L. Miki T. Rosenberg S.A. Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor.Proc. Natl. Acad. Sci. USA. 1994; 91 (a): 3515-3519Crossref PubMed Scopus (1009) Google Scholar). The immunodominance of this antigen in HLA-A2 individuals is striking (22Kawakami Y. Eliyahu S. Sakaguchi K. Robbins P.F. Rivoltini L. Yannelli J.R. Appella E. Rosenberg S.A. Identification of the immunodominant peptides of the MART-1 human melanoma antigen recognized by the majority of HLA-A2 restricted tumor infiltrating lymphocytes.J. Exp. Med. 1994; 180 (c): 347-352Crossref PubMed Scopus (748) Google Scholar). Of 29 HLA-A2+ TIL exhibiting HLA-A2-restricted recognition of shared tumor antigens, 21 reacted with the MART-1 protein. In all 21 cases, these TIL recognized the identical 9-mer AAGIGILTV peptide. A MART-1 epitope has also been identified that is restricted by HLA-B45 (52Schneider J. Brichard V. Boon T. Overlapping peptides of melanocyte differentiation antigen Melan-A/MART-1 recognized by autologous cytolytic T lymphocytes in association with HLA-B45.1 and HLA-A2.1.Int. J. Cancer. 1998; 75: 451-458Crossref PubMed Scopus (48) Google Scholar). Responses to the MART-1/MelanA epitope can be readily elicited in vitro from peripheral lymphocytes of normal nonmelanoma-containing individuals (33Marincola F.M. Rivoltini L. Salgaller M.L. Player M. Rosenberg S.A. Differential anti-MART-1/MelanA CTL activity in peripheral blood of HLA-A2 melanoma patients in comparison to healthy donors evidence for in vivo priming by tumor cells.J. Immunother. 1996; 19: 266-277Crossref Scopus (118) Google Scholar). MART-1-reactive T cells can react with differing peptides derived from a variety of exogenous antigens including viral proteins, and cross-reactivity with these epitopes may result in the high precursor frequency of T cells against the MART-1 epitope (29Loftus D.J. Appella E. Rivoltini L. Identification of epitope mimics recognized by CTL reactive to the melanoma/melanocyte-derived peptide MART-1 27–35.J. Exp. Med. 1996; 184: 647-657Crossref PubMed Scopus (151) Google Scholar). Of the 29 HLA-A2-reactive TIL, 13 reacted with the gp100 melanoma differentiation antigen (21Kawakami Y. Eliyahu S. Delgado C.H. Robbins P.F. Sakaguchi K. Appella E. Yannelli J.R. Adema G.J. Miki T. Rosenberg S.A. Identification of a human melanoma antigen recognized by tumor infiltrating lymphocytes associated with in vivo tumor rejection.Proc. Natl. Acad. Sci. USA. 1994; 91 (b): 6458-6462Crossref PubMed Scopus (841) Google Scholar). This 661–amino acid protein was identified independently by screening cDNA expression libraries with melanoma reactive lymphocytes (21Kawakami Y. Eliyahu S. Delgado C.H. Robbins P.F. Sakaguchi K. Appella E. Yannelli J.R. Adema G.J. Miki T. Rosenberg S.A. Identification of a human melanoma antigen recognized by tumor infiltrating lymphocytes associated with in vivo tumor rejection.Proc. Natl. Acad. Sci. USA. 1994; 91 (b): 6458-6462Crossref PubMed Scopus (841) Google Scholar) as well as by elution of peptides from an HLA-A2+ melanoma (11Cox A.L. Skipper J. Chen Y. Henderson R.A. Darrow T.L. Shabanowitz J. Engelhard V.H. Hunt D.F. Slingluff C.L. Identification of a peptide recognized by five melanoma-specific human cytotoxic T cell lines.Science. 1994; 264: 716-719Crossref PubMed Scopus (799) Google Scholar). This gene had previously been isolated and was known to encode a protein recognized by the HMB-45 monoclonal antibody but was unknown as a T cell antigen. The gp100 protein encodes an enzyme involved in melanin synthesis, and multiple gp100 epitopes have been identified restricted not only by HLA-A2 but by HLA-A3 and HLA-A24 as well (23Kawakami Y. Eliyahu S. Jennings C. Sakaguchi K. Kang X. Southwood S. Robbins P.F. Sette A. Appella E. Rosenberg S.A. Recognition of multiple epitopes in the human melanoma antigen gp100 by tumor infiltrating T-lymphocytes associated with in vivo tumor regression.J. Immunol. 1995; 154: 3461-3968PubMed Google Scholar, 57Skipper J.C. Kittlesen D.J. Hendrickson R.C. Deacon D.D. Harthun N.L. Wagner S.N. Hunt D.F. Engelhard V.H. Slingluff Jr., C.L. Shared epitopes for HLA-A3-restricted melanoma-reactive human CTL include a naturally processed epitope from Pmel-17/gp100.J. Immunol. 1996; 157 (b): 5027-5033PubMed Google Scholar, 42Robbins P.F. El-Gamil M. Li Y.F. The intronic region of an incompletely spliced gp100 gene transcript encodes an epitope recognized by melanoma-reactive tumor-infiltrating lymphocytes.J. Immunol. 1997; 159: 303-308PubMed Google Scholar, 63Tsai V. Southwood S. Sidney J. Identification of subdominant CTL epitopes of the gp100 melanoma-associated tumor antigen by primary in vitro immunization with peptide-pulsed dendritic cells.J. Immunol. 1997; 158: 1796-1802PubMed Google Scholar, 24Kawakami Y. Robbins P.F. Wang X. Tupesis J.P. Parkhurst M.R. Kang X. Sakaguchi K. Appella E. Rosenberg S.A. Identification of new melanoma epitopes on melanosomal proteins recognized by tumor infiltrating T lymphocytes restricted by HLA-A21, -A2, and -A3 alleles.J. Immunol. 1998; 161: 6985-6992PubMed Google Scholar). Interestingly, the epitope recognized by HLA-A24-restricted T cells results from an aberrantly spliced product of the gp100 gene, resulting in translation of the fourth intron of the gp100 molecule in both melanomas and in normal melanocytes (42Robbins P.F. El-Gamil M. Li Y.F. The intronic region of an incompletely spliced gp100 gene transcript encodes an epitope recognized by melanoma-reactive tumor-infiltrating lymphocytes.J. Immunol. 1997; 159: 303-308PubMed Google Scholar). Tyrosinase, an enzyme critical for the synthesis of melanin, has been shown to be an antigen recognized by HLA-A1-, HLA-A2-, and HLA-A24-restricted T cells (5Brichard V. Van Pel A. Wolfel T. Wolfel C. De Plaen E. Lethe B. Coulie P. Boon T. The tyrosinase gene codes for an antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas.J. Exp. Med. 1993; 178: 489-495Crossref PubMed Scopus (892) Google Scholar, 39Robbins P.F. El-Gamil M. Kawakami Y. Rosenberg S.A. Recognition of tyrosinase by tumor infiltrating lymphocytes from a patient responding to immunotherapy.Cancer Res. 1994; 54: 3124-3126PubMed Google Scholar, 78Wolfel T. Van Pel A. Brichard V. Schneider J. Seliger B. Meyer zum Buschenfeld K.-H. Boon T. Two tyrosinase nonapeptides recognized on HLA-A2 melanomas by autologous cytolytic T lymphocytes.Eur. J. Immunol. 1994; 24: 759-764Crossref PubMed Scopus (379) Google Scholar, 25Kittlesen D.J. Thompson L.W. Gulden P.H. Human melanoma patients recognize an HLA-A1-restricted CTL epitope from tyrosinase containing two cysteine residues implications for tumor vaccine development.J. Immunol. 1998; 160: 2099-2106PubMed Google Scholar). One of the naturally occurring peptide epitopes restricted by HLA-A2 contains a posttranslational modification of an asparagine to aspartic acid residue (56Skipper J.C.A. Hendrickson R.C. Gulden P.H. Brichard V. Van Pel A. Chen Y. Shabanowitz J. Wolfel T. Slingluff C.L. Boon T. et al.An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins.J. Exp. Med. 1996; 183 (a): 527-534Crossref PubMed Scopus (368) Google Scholar). Tyrosinase is the only melanoma/melanocyte differentiation antigen known to be recognized by CD4+ tumor-reactive T cells (61Topalian S.L. Rivoltini L. Mancini M. Markus N.R. Robbins P.F. Kawakami Y. Rosenberg S.A. Human CD4+ T cells specifically recognize a shared melanoma-associated antigen encoded by the tyrosinase gene.Proc. Natl. Acad. Sci. USA. 1994; 91: 9461-9465Crossref PubMed Scopus (263) Google Scholar). A CD4+ tumor-infiltrating lymphocyte cross-reactive with HLA-DR0401+ melanomas was used to test for reactivity against candidate melanoma antigens previously shown to be recognized by CD8+ T cells, and tyrosinase was identified as the reactive antigen. Several class II–presented epitopes from tyrosinase have also been identified. Two melanocyte proteins that bear approximately 40% amino acid homology to tyrosinase have been identified as T cell antigens (72Wang R.-F. Robbins P.F. Kawakami Y. Kang X.Q. Rosenberg S.A. Identification of a gene encoding a melanoma tumor antigen recognized by HLA-A31-restricted tumor-infiltrating lymphocytes.J. Exp. Med. 1995; 181: 799-804Crossref PubMed Scopus (268) Google Scholar, 73Wang R.-F. Parkhurst M.R. Kawakami Y. Robbins P.F. Rosenberg S.A. Utilization of an alternative open reading frame of a normal gene in generating a novel human cancer antigen.J. Exp. Med. 1996; 183: 1131-1140Crossref PubMed Scopus (213) Google Scholar, 74Wang R.-F. Johnston S.L. Southwood S. Sette A. Rosenberg S.A. Recognition of an antigenic peptide derived from tyrosinase-related protein-2 by CTL in the context of HLA-A31 and -A33.J. Immunol. 1998; 160 (a): 890-897PubMed Google Scholar). TRP-1 is the most prevalent protein present in melanocytes and melanomas and was recognized as a tumor antigen reactive with HLA-A31-restricted TIL 586 from a patient with melanoma. The peptide epitope reactive with TIL 586 was encoded by the normal TRP-1 gene but was not translated from the primary open reading frame (73Wang R.-F. Parkhurst M.R. Kawakami Y. Robbins P.F. Rosenberg S.A. Utilization of an alternative open reading frame of a normal gene in generating a novel human cancer antigen.J. Exp. Med. 1996; 183: 1131-1140Crossref PubMed Scopus (213) Google Scholar). The third open reading frame of this gene translated a 21–amino acid peptide that contained the 9–amino acid peptide epitope recognized by TIL 586. This alternative open reading frame was also translated in normal melanocytes. At least one other example of an alternative open reading frame encoding a tumor antigen was subsequently described (75Wang R.-F. Johnston S.L. Topalian S.L. Schwartzentruber D.J. Rosenberg S.A. A breast and melanoma-shared tumor antigen T cell responses to antigenic peptides translated from different open reading frames.J. Immunol. 1998; 161 (b): 3596-3606Google Scholar), and rare other examples exist of the recognition of the products of alternative open reading frames (30Malarkannan S. Afkarian M. Shastri N. A rare cryptic translation product is presented by Kb major histocompatibility complex class I molecule to alloreactive T cells.J. Exp. Med. 1995; 182: 1739-1750Crossref PubMed Scopus (62) Google Scholar, 38Quelle D.E. Zindy F. Ashmun R.A. Sherr C.J. Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest.Cell. 1995; 83: 993-1000Abstract Full Text PDF PubMed Scopus (1275) Google Scholar, 12Elliott T. Bodmer H. Townsend A. Recognition of out-of-frame major histocompatibility complex class I-restricted epitopes in vivo.Eur. J. 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Definition of an HLA-A3-like super motif demonstrates the overlapping peptide-binding repertoires of common HLA molecules.Hum. Immunol. 1996; 45: 79-93Crossref PubMed Scopus (190) Google Scholar, 74Wang R.-F. Johnston S.L. Southwood S. Sette A. Rosenberg S.A. Recognition of an antigenic peptide derived from tyrosinase-related protein-2 by CTL in the context of HLA-A31 and -A33.J. Immunol. 1998; 160 (a): 890-897PubMed Google Scholar). Studies of the TRP-2 protein represent an example of the use of in vitro sensitization techniques to identify new tumor antigenic epitopes. To identify HLA-A2-restricted TRP-2 epitopes, multiple peptides were synthesized from the TRP-2 protein based on HLA-A2-binding motifs. Using in vitro sensitization techniques against those multiple peptides, an HLA-A2-restricted T cell was generated against a single TRP-2 peptide, SVYDFFVWL, that was also capable of recognizing HLA-A2+ melanomas (37Parkhurst M.R. Fitzgerald E.B. Southwood S. Sette A. Rosenberg S.A. Kawakami Y. Identification of a shared HLA-A*0201-restricted T-cell epitope from the melanoma antigen tyrosinase-related protein 2 (TRP2).Cancer Res. 1998; 58: 4895-4901PubMed Google Scholar). Thus, at least five melanoma/melanocyte differentiation proteins have been shown to mediate the generation of T cells capable of recognizing melanomas restricted by multiple class I alleles. Another class of shared differentiation antigens is expressed on tumors as well as germ cells of the testes. The first cloned human tumor antigen recognized by T cells was isolated utilizing T cells from a melanoma patient who had been repeatedly immunized with mutagenized cancer cells (68Van der Bruggen P. Traversari C. Chomez P. Lurquin C. DePlaen E. Van Den Eynde B. Knuth A. Boon T. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma.Science. 1991; 254: 1643-1647Crossref PubMed Scopus (3004) Google Scholar). The T cells from this individual were sensitized in vitro to the autologous melanoma, and reactive T cell clones that recognized a protein encoded by a gene termed MAGE-1 were identified. This gene belonged to a multigene family containing at least 12 different genes expressed in a small percentage of melanomas as well as cancers arising in the breast, prostate, esophagus, colon, and lung (4Brasseur F. Marchand M. Vanwijck R. Herin M. Lethe B. Chomez P. Boon T. Human gene MAGE-1, which codes for a tumor-rejection antigen, is expressed by some breast tumors. Letter to the editor.Int. J. Cancer. 1992; 52: 839-841Crossref PubMed Scopus (164) Google Scholar, 62Traversari C. Van der Bruggen P. Luescher I.F. Lurquin C. Chomez P. Van Pel A. De Plaen E. Amar-Costesec A. Boon T. A nonapeptide encoded by human gene MAGE-1 is recognized on HLA-A1 by cytolytic T lymphocytes directed against tumor antigen MZ2-E.J. Exp. Med. 1992; 176: 1453-1457Crossref PubMed Scopus (623) Google Scholar, 2Boon T. 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This family of tumor antigens was detected using in vitro sensitized T cells from the autologous patient, although no descriptions of TIL recognizing these antigens have yet been reported. The levels of MAGE-1 expressed by some tumor cells can be below the level of detection required for lymphocyte recognition (28Lethe B. Van der Bruggen P. Brasseur F. Boon T. MAGE-1 expression threshold for the lysis of melanoma cell lines by a specific cytotoxic T lymphocyte.Melanoma Res. 1997; 2: S83-S88Google Scholar), even though some studies suggest that as few as five peptides on the cell surface are sufficient for T cell recognition (6Brower R.C. England R. Takeshita T. Minimal requirements for peptide mediated activation of CD8+ CTL.Mol. Immunol. 1994; 31: 1285-1293Crossref PubMed Scopus (68) Google Scholar, 59Sykulev Y. Joo M. Vturina I. Tsomides T.J. Eisen H.N. Evidence that a single peptide-MHC complex on a target cell can elicit a cytolytic T cell response.Immunity. 1996; 4: 565-571Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar). Another antigen expressed in cancers and testes is t